Valerie Weaver, PhD
Co-Principal Investigator and Leader of Project 3
Introduction to Project 3
The mortality associated with late-stage glioblastoma (GBM) can be explained by their early dissemination and intrinsic death resistance. Magnetic resonance imaging (MRI) suggests that poor-prognosis GBM that arise in the sunbventricular zone (SVZ) are highly vascularized and have a high cellular content, along with an abundant extracellular matrix (ECM) that promotes perfusion and impedes diffusion, resulting in an increase in intracranial pressure. A high-force environment also enhances the secretion of chemokines and growth factors and potentiates their signaling to drive the growth and invasion of GBM, as well as to promote tissue inflammation and angiogenesis. Furthermore, such an environment favors the growth and survival of flexible (compliant), contractile and mechanically-resistant GBM, because these traits facilitate their expansion and survival within a confined space and foster their ability to navigate through the dense ECM. These characteristics are consistent with a neural stem cell (NSC) origin. In fact, we found that NSC and GBM have high NCoR2, which induces pleiotropic treatment resistance. We also determined that NSC and late stage GBM are softer (more compliant) than early stage oligodendro tumors. This, combined with the fact that GBM have elevated active FAK and an altered glycocalyx and spread more on soft ECM, indicates that GBM are death resistant and have an altered mechano-phenotype.
In this proposal we will test the prediction that the mechanically-challenged landscape of aggressive GBM and their unique mechano-phenotype favor their growth, survival and invasion. We will test this hypothesis by addressing three major aims. In Aim 1, we will test the idea that GBM aggression is associated with a mechanically-challenged SVZ brain microenvironment and a unique mechano-phentoype. MRI, atomic force microscopy (AFM), and immunohistochemistry (IHC) will be used to build a spatial map of the mechanical landscape of human and mouse GBM in the SVZ region as related to histology, aggression and stemness. AFM, TFM and cellular approaches will be used to quantify the mechano-phenotype of GBM, and compression and ECM stiffness will be varied to assess their mechano-responsiveness.
In Aim 2, we will test the hypothesis that the mechano-phenotype and aggressive behavior of GBM is mediated by hyaluronan (HA)-induced changes in the glycocalyx, integrins and Notch. In turn, this will identify a new paradigm with which to understand the role of an abundant ECM protein HA in GBM biology; define a new mechanism whereby GBM evade treatment; implicate force as a key regulator of stemness; and develop a simple strategy to identify treatment-resistant GBM in biopsies. Scanning-angle fluorescence interference (SA-FLIC) imaging and cell biological approaches will assess the adhesion and glycocaylx behavior of GBM (Aim 1), and gain and loss of function studies will test functional links among HA, integrins, Notch and GBM aggression both in vitro and in vivo.
Finally, in Aim 3 we will test the proposition that tissue force and the GBM mechano-phenotype foster the vascular niche. We will assay the effect of force on GBM-derived inflammatory and angiogenic factors and identify plausible mechano-transduction mechanisms. We all also establish functional links through orthotopic intracranial implantation studies and a series of transgenic crosses. Because of their ready availability, we will first conduct experiments using the human glioma lines characterized in Aim 1. Once the b1 and FAK knockdown, V737N knockin b1 integrin and activated ROCK mice become available, we will isolate murine NSC and transform them with V12HaRas or a constitutively active EGFR and conduct SVZ intracranial orthotopic implant studies. The orthotopic manipulations will be complemented through a series of mouse crosses in which we will assess the consequence of modulating integrin adhesion and ROCK activity on GFAPV12Ha-Ras induced GBM tumor formation, GBM morphology, growth, vascularity and inflammation. These studies will be conducted in collaboration with Dr. Bergers' group.
The clinical studies in Aim 1 will initiate in Year 1 and continue for the duration of the project. Aim 1 animal studies will commence in Year 1 and continue until Year 3, while cell line studies will begin in Year 1 and should finish within two years. For Aim 2, SA-FLIC imaging of clinical samples will start toward the end of Year 1 and continue for the duration of the project. The cell line work and mechanistic studies will initiate at the end of Year 1 and should be completed by the middle to end of Year 4. Meanwhile, the GBM transplant studies will commence in the middle to end of Year 2 and continue until the end of the project. The in vitro studies in Aim 3 will commence in the middle of Year 1 and should be completed by the end of Year 2, while the GBM transplant studies will commence by the beginning of Year 3 and continue through the end of the project. The transgenic crosses will begin immediately and continue until the end of the project, although we do not expect to have cohorts for study until Year 3.
Image Compliments of Matthew Paszek, Post-Doc and Yakaterina Miroshnikova, Graduate Student